Modulation of phosphofructokinase (PFK) from Setaria cervi, a bovine filarial parasite, by different effectors and its interaction with some antifilarials

Abstract

Background: Phosphofructokinase (ATP: D-fructose-6-phosphate-1-phosphotransferase, EC 2.7.1.11, PFK) is of primary importance in the regulation of glycolytic flux. This enzyme has been extensively studied from mammalian sources but relatively less attention has been paid towards its characterization from filarial parasites. Furthermore, the information about the response of filarial PFK towards the anthelmintics/antifilarial compounds is lacking. In view of these facts, PFK from Setaria cervi, a bovine filarial parasite having similarity with that of human filarial worms, was isolated, purified and characterized.

Results: The S. cervi PFK was cytosolic in nature. The adult parasites (both female and male) contained more enzyme activity than the microfilarial (Mf) stage of S. cervi, which exhibited only 20% of total activity. The S. cervi PFK could be modulated by different nucleotides and the response of enzyme to these nucleotides was dependent on the concentrations of substrates (F-6-P and ATP). The enzyme possessed wide specificity towards utilization of the nucleotides as phosphate group donors. S. cervi PFK showed the presence of thiol group(s) at the active site of the enzyme, which could be protected from inhibitory action of para-chloromercuribenzoate (p-CMB) up to about 76% by pretreatment with cysteine or β-ME. The sensitivity of PFK from S. cervi towards antifilarials/anthelmintics was comparatively higher than that of mammalian PFK. With suramin, the Ki value for rat liver PFK was 40 times higher than PFK from S. cervi.

Conclusions: The results indicate that the activity of filarial PFK may be modified by different effectors (such as nucleotides, thiol group reactants and anthelmintics) in filarial worms depending on the presence of varying concentrations of substrates (F-6-P and ATP) in the cellular milieu. It may possess thiol group at its active site responsible for catalysis. Relatively, 40 times higher sensitivity of filarial PFK towards suramin as compared to the analogous enzyme from the mammalian system indicates that this enzyme could be exploited as a potential chemotherapeutic target against filariasis.

Figure 1

Effect of cAMP on the rate of PFK catalyzed reaction under inhibitory (A and B) and optimal (C and D) substrate concentrations. Figure 1A. Effect of cAMP on the rate of S. cervi PFK catalyzed reaction at fixed inhibitory concentration of ATP (1.0 mM) and low concentration of F-6-P (0.5 mM). Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 6.6 μg/ml. Other conditions were the same as in standard enzyme assay. V and V₀ are rates of reaction in the presence and absence of cAMP. Figure 1B. Double reciprocal plot of the data of Figure 1A. Figure 1C. Effect of cAMP on the rate of S. cervi PFK catalyzed reaction at optimal concentrations of substrates (F-6-P, 3.3 mM; ATP, 0.10 mM). Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 6.6 μg/ml. Other conditions were the same as in standard enzyme assay. V and V₀ are rates of reaction in the presence and absence of cAMP. Figure 1D. Double reciprocal plot of the data of Figure 1C.

Figure 2

Effect of AMP on the rate of PFK catalyzed reaction under inhibitory (A and B) and optimal (C) substrate concentrations. Figure 2A. Effect of AMP on the rate of S. cervi PFK catalyzed reaction at fixed inhibitory concentration of ATP (1.0 mM) and low concentration of F-6-P (0.5 mM). Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 6.6 μg/ml. Other conditions were the same as in standard enzyme assay. V and V₀ are rates of reaction in the presence and absence of AMP. Figure 2B. Double reciprocal plot of the data of Figure 2A. Figure 2C: Effect of AMP on the rate of S. cervi PFK catalyzed reaction at optimal concentrations of substrates (F-6-P, 3.3 mM; ATP, 0.10 mM). Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 10.0 μg/ml. Other conditions were the same as in standard enzyme assay.

Figure 3

Effect of ADP on the rate of PFK catalysed reaction under inhibitory (A and B) and optimal (C and D) substrate concentrations. Figure 3A. Effect of ADP on the rate of S. cervi PFK catalyzed reaction at fixed inhibitory concentration of ATP (1.0 mM) and low concentration of F-6-P (0.5 mM). Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 6.6 μg/ml. Other conditions were the same as in standard enzyme assay. V and V₀ are rates of reaction in the presence and absence of ADP. Figure 3B. Double reciprocal plot of the data of Figure 3A. Figure 3C. Effect of ADP on the rate of S. cervi PFK catalyzed reaction at optimal concentrations of substrates (F-6-P, 3.3 mM; ATP, 0.10 mM). Mg²⁺ concentration was constant (3.3 mM). Enzyme concentration was 6.6 μg/ml. Other conditions were the same as in standard enzyme assay. V and V₀ are rates of reaction in the presence and absence of ADP. Figure 3D. Double reciprocal plot of the data of Figure 3C.

Figure 4

Effect of suramin on the rate of S. cervi PFK-catalyzed reaction (A) and the Hill plot of the data presented in (A). Figure 4A. Effect of suramin on the rate of S. cervi PFK-catalyzed reaction. Suramin concentrations were nil (○), 1 (Δ) and 5 nM (●). Mg²⁺ concentration was constant (3.3 mM). The concentration of ATP was 0.1 mM. Enzyme concentration used was 10 μg/ml. Other conditions were same as described in Materials and Methods. Figure 4B. Hill plot of the data on the effect of suramin (0-10 nM) on the rate of S. cervi PFK-catalyzed reaction. The concentrations of F-6-P, ATP and Mg²⁺ were 3.3, 0.1 and 3.3 mM. Other conditions were same as described in Materials and Methods. V_i and V₀ are rates of reaction in the presence and absence of suramin.

Figure 5

Effect of suramin on the rate of rat liver PFK-catalyzed reaction (A) and Hill plot (B) of the data presented in (A). Figure 5A. Effect of suramin on the rate of rat liver PFK-catalyzed reaction. Suramin concentrations were nil (○), 1 (Δ) and 5 nM (●). Mg²⁺ concentration was constant (3.3 mM). The concentration of ATP was 0.1 mM. Enzyme concentration used was 10 μg/ml. Other conditions were same as described in Materials and Methods. Figure 5B. Hill plot of the data on the effect of suramin (0-50 nM) on the rate of rat liver PFK-catalyzed reaction. The concentrations of F-6-P, ATP and Mg²⁺ were 3.3, 0.1 and 3.3 mM. Other conditions were the same as described in Materials and Methods. V_i and V₀ are rates of reaction in the presence and absence of suramin.

Figure 6

Coupled enzyme assay system. Schematic presentation of the biochemical reactions taking place during assay of the activity of S. cervi PFK. It is a coupled enzyme assay system in which the auxiliary enzymes have been used in excess so as to achieve overall rate of reaction being governed by PFK.